Injured tissues are regenerated by the following process: removal of useless tissue fragments and cell fragments or bacteria and so on by phagocytes such as macrophages that migrate to the injured site;
recovery of vessels; and the subsequent tissue renewal. Transforming growth factor β(TGF-β) produced by macrophages and neutrophils, which appear during the process of the tissue regeneration and recovery, has been revealed to serve as the first regulatory factor in the regeneration-recovery process.
TGF-β has multiple functions. The factor is known to regulate the production of the extracellular matrix (ECM) from connective tissue cells as well as to induce the proliferation of mesenchymal cells and to inhibit the proliferation of vascular endothelial cells and epithelial cells.
Increased production of platelet-derived growth factor (PDGF) and connective tissue growth factor (CTGF; also called Hcs24) is found in the culture supernatant of the above-mentioned mesenchymal cells of which proliferation is induced by a stimulus with TGF-β. Because of this, it is presumed that the cell proliferation is not directly but indirectly induced by TGF-β with the help of other regulatory factors.
Human and mouse CTGFs have been identified previously (so far, there is no report on the identification of rat CTGF), and their physicochemical and biological properties have been analyzed (<human CTGF>: J. Cell Biology, vol. 114, No. 6, p. 1285-1294, 1991; Int. J. Biochem. Cell Biol., Vol. 29, No. 1, p. 153-161, 1997; Circulation, vol. 95, No. 4, p. 831-839, 1997; Cell Growth Differ., Vol. 7, No. 4, p. 469-480, 1996; J. Invest. Dermatol., Vol. 106, No. 4, p. 729-733, 1996; J. Invest. Dermatol., Vol. 105, No. 2, p. 280-284, 1995; J. Invest. Dermatol. Vol. 105, No. 1, p. 128-132, 1995; WO96/38172; <mouse CTGF (Fisp12)>: Unexamined Published Japanese Patent Application (JP-A) No. Hei 5-255397; Cell Growth Differ., vol. 2, No. 5, p. 225-233, 1991; FEBS Letters, Vol. 327, No. 2, p. 125-130, 1993; DNA Cell Biol., Vol. 10, No. 4, p. 293-308, 1991).
CTGF is a cysteine-rich secretory glycoprotein with a molecular weight of about 38 kDa. It has been revealed that the biosynthesis and secretion of the protein are induced by TGF-β. CTGF has similar properties with PDGF in the light of that: their productions are induced by TGF-β; they bind to the PDGF receptor and induce the proliferation of mesenchymal cells; and they are produced by fibroblasts and epithelial cells. However, they exhibit no homology at the amino acid level and thus the two molecules are distinct to each other (The Journal of Cell Biology, vol. 114, No. 6, p. 1287-1294, 1991; Molecular Biology of the Cell, Vol. 4, p. 637-645, 1993).
In recent studies, low molecular weight species of CTGF have been found in the culture supernatant of human and mouse fibroblast cells as well as in the secreting fluid derived from the porcine uterus. They are biologically active but are presumed to be degradation products of 38 kDa CTGF molecules, since their molecular weights are about 10-12 kDa (Growth Factors, vol. 15, No. 3, p. 199-213, 1998; J. Biol. Chen., vol. 272, No. 32, p. 20275-20282, 1997).
Details of the relationship between physiological functions of CTGF and diseases have yet to be fully clarified. However, it has been found that: CTGF production is induced by TGF-β; the expression level of CTGF mRNA is significantly high in tissues and cells derived from patients affected with various diseases (Int. J. Biochem. Cell. Biol., Vol. 29, No. 1, p. 153-161, 1997; Circulation, Vol. 95, No. 4, p. 831-839, 1997; J. Invest. Dermatol, Vol. 106, No. 4, p. 729-733, 1996; J. Invest. Dermatol., Vol. 105, No. 2, p. 128-132, 1995; J. Cell Physiol., Vol. 165, No. 3, p. 556-565, 1995; Kidney Int., Vol. 48, No. 2, p. 5001-5009, 1995); and CTGF enhances the chemotaxis and proliferation of the vascular endothelial cells (J. Cell. Biol., Vol. 114, No. 6, p. 1285-1294, 1991; Exp. Cell Res., Vol. 233, p. 63-77, 1997; Journal of Japanese Association for Oral Biology, Vol. 38, extra number, p. 463, PD0187, 1996; the 69th meeting of the Japanese Biochemical Society, proceedings, p. 683, 1P0535, 1996). These findings suggest the possibility that CTGF is associated with the onset and/or advancement of a variety of diseases.
Identification of the specific diseases awaits further findings and advancement in research. Nonetheless, CTGF has been presumed to be involved in the onset and/or advancement of a wide variety of diseases including, for example, cancers, arteriosclerosis, and skin diseases (for example, psoriasis, scleroderma, atopy, and keloid), kidney diseases, arthritis (for example, rheumatoid arthritis), various fibrotic diseases (fibrotic diseases in tissues as observed in arteriosclerosis, cirrhosis, arthritis, scleroderma, keloid, kidney fibrosis and pulmonary fibrosis, etc.).
To elucidate the association of CTGF with such various diseases, it is generally effective to detect and assay CTGF and/or the protein fragments thereof in the body fluids (serum, etc.) from patients and mammals affected with the diseases; the values determined are compared with normal values (obtained from mammals including normal persons, normal mice, normal rats and normal rabbits, etc.).
The detection and assay of secretory proteins such as CTGF are carried out by immunological assays based on antigen-antibody interaction by using the antibody (preferably used are monoclonal antibodies) which is reactive to the secretory protein to be detected; specifically, immunoassays such as radioimmunoassay (RIA) and enzyme immunoassay (EIA, ELISA) are widely used as the most convenient and useful methods for the purpose.
In this context, for the purpose of assaying CTGF, it is necessary to develop detection and assay methods using such immunoassay systems and also to prepare monoclonal antibodies against CTGF required for the establishment of assay methods. There are some reports on the preparation of antiserum reactive to CTGF (Exp. Cell Res., Vol. 233, p. 63-77, 1997; Cell Growth Differ., Vol. 8, No. 1, p. 61-68, 1997; the 69th meeting of Japanese Biochemical Society, proceedings, p. 683, 1P0534, 1996) but no report on the preparation of functional anti-CTGF monoclonal antibody which has particularly high affinity for CTGF and/or the capability of neutralizing the CTGF activity; no immunoassay systems for CTGF have so far been provided at all.
Such monoclonal antibodies having the capability of neutralizing the CTGF activity described hereinabove are useful not only as components in an immunoassay system but also as pharmaceutical antibody preparations for the treatment and/or prevention of the above-mentioned diseases caused by CTGF secretion. However, there have not been any report on such monoclonal antibodies yet.